Download Cell surface monoamine oxidases: enzymes in search of a function

The EMBO Journal Vol. 20 No. 15 pp. 3893±3901, 2001
NEW EMBO MEMBER'S REVIEW
Cell surface monoamine oxidases: enzymes in
search of a function
S.Jalkanen1 and M.Salmi
MediCity Research Laboratory, Turku University, TykistoÈkatu 6A,
20520 Turku and National Public Health Institute, Department in
Turku, Turku, Finland
1
Corresponding author
e-mail: sirpa.jalkanen@utu.®
Ectoenzymes with a catalytically active domain
outside the cell surface have the potential to regulate
multiple biological processes. A distinct class of
copper-containing semicarbazide-sensitive monoamine
oxidases, expressed on the cell surface and in soluble
forms, oxidatively deaminate primary amines. Via
transient covalent enzyme±substrate intermediates,
this reaction results in production of aldehydes,
hydrogen peroxide and ammonium, which are all
biologically active substances. The physiological functions of these enzymes have remained unknown,
although they have been suggested to be involved in
the metabolism of biogenic amines. Recently, new
roles have been proposed for these enzymes in regulation of glucose uptake and, even more surprisingly, in
leukocyte±endothelial cell interactions. The emerging
functions of ectoenzymes in signalling and cell±cell
adhesion suggest a novel mode of molecular control of
these complex processes.
Keywords: cell adhesion/leukocyte traf®cking/oxygen
radicals/signalling/SSAO
Introduction
Enzymes have been traditionally thought to act as catalysts
in complex biochemical pathways within a cell, and they
have been classi®ed according to the nature of the
enzymatic reaction they catalyse (e.g. nucleotidases,
oxidases, kinases). This functional division (rather than
sequence alignments) also forms the basis for the current
Enzyme Commission (EC) classi®cation (http://www.expasy.ch/enzyme/enzyme_details.html). Recently, an
increasing number of enzymes are being recognized as
cell surface proteins (e.g. Goding and Howard, 1998).
These molecules have the capacity to catalyse enzymatic
reactions in the immediate vicinity of the cell surface, and
thereby regulate the concentration and functions of their
substrates and end products, which are often biologically
active. Consequently, ectoenzymes have been implicated
in uptake and recycling of nutrients and in degradation of
extracellular foreign DNA. They can also regulate cell
signalling by producing or destroying nucleotides (e.g.
CD73/Ecto-5¢ nucleotidase dephosphorylates AMP and
IMP to adenosine and inosine, respectively) or by locally
activating or inactivating biologically active peptides (e.g.
CD143/Angiotensin-converting enzyme is responsible for
ã European Molecular Biology Organization
proteolytic cleavage of inactive angiotensin I to biologically active angiotensin II). However, these cell surface
enzymes are often large glycoproteins themselves and
hence can provide many protein domains and oligosaccharide moieties for other non-enzymatic recognition
events as well. Here we summarize some of the data that
have accumulated during recent years to reveal the
biological function of semicarbazide-senistive amine
oxidases (SSAOs), one special class of ectoenzymes.
Enzymatic classi®cation of amine oxidases
Amine oxidases (AOs) have been traditionally divided into
two main groups, based on the chemical nature of the
attached cofactor (Figure 1A). The ¯avin adenine
dinucleotide (FAD)-containing enzymes [monoamine
oxidase A (MAO-A), MAO-B and polyamine oxidases]
are intracellular enzymes (Shih et al., 1999). The other
class of AOs contain a cofactor possessing one or more
carbonyl groups, which appears to be topa-quinone (TPQ)
in most cases (Klinman and Mu, 1994; Klinman, 1996;
Lyles, 1996). These enzymes include diamine oxidases,
lysyl oxidase, and plasma membrane and soluble MAOs.
These enzymes are collectively designated as SSAOs due
to their characteristic sensitivity of inhibition by a
carbonyl-reactive compound, semicarbazide.
The FAD- and TPQ-containing AOs not only differ in
their cofactors, but are also distinct in terms of their
subcellular distribution, substrates, inhibitors and biological functions (Figure 1). MAO-A and -B are well
known mitochondrial enzymes that have ®rmly established roles in the metabolism of neurotransmitters (e.g.
noradrenaline) and other biogenic amines (e.g. tyramine,
adrenaline) (Shih et al., 1999). FAD-containing polyamine
oxidases use secondary amines spermine and spermidine
as their preferred substrates, and thereby possibly regulate
cell growth (Seiler, 1990).
On the other hand, the TPQ-containing enzyme diamine
oxidase prefers diamines putrescine and cadaverine as its
substrate (Buffoni, 1966; Robinson-White et al., 1985;
Barbry et al., 1990). Diamine oxidase is mainly an
intracellular enzyme preferentially synthesized in the
placenta, kidney and intestine. The secreted form binds
in a heparin-dependent manner to endothelial cells.
Diamine oxidase also oxidizes histamine, and hence is
important in regulating in¯ammation and allergic reactions. In fact, it was ®rst described as an amiloride binding
protein believed to function as a sodium channel in human
kidney (Barbry et al., 1990), and only later found to be a
semicarbazide-sensitive diamine oxidase (Mu et al., 1994;
Novotny et al., 1994).
The other topa-containing SSAOs are mostly soluble or
expressed on the cell surface, have different preferred
substrates, are insensitive or only weakly sensitive to
3893
S.Jalkanen and M.Salmi
3894
Cell surface monoamine oxidases
classical MAO inhibitors (like chlorgyline or deprenyl),
and mediate different biological functions (Lyles, 1996).
The rest of this review will only discuss these extracellular
MAOs. Moreover, although these SSAOs are present in
bacteria, yeasts and plants, as well as in higher eukaryotes,
we will restrict the discussion mainly to the mammalian
enzymes.
SSAOs have been historically de®ned by their inhibition
with carbonyl-reactive compounds like semicarbazide
(Tabor et al., 1954). Thereafter, multiple other SSAO
inhibitors have been described (reviewed in Lyles, 1996).
Notably, a simple compound hydroxylamine (NH2-OH) is
a more selective and potent SSAO inhibitor. Various other
compounds, including propargylamine (acetylenic aliphatic amine), aminoguanidine (nitric oxide synthase inhibitor), carbidopa (DOPA decarboxylase inhibitor) and
procarbazine (carcinostatic agent) are also inhibitors of
SSAOs, although far from speci®c. Interestingly, several
drugs in clinical use (e.g. an anti-microbe isoniazid, antihypertensive hydralazine, anti-arrythmic mexiletine) also
inhibit SSAOs. In most cases, however, the contribution of
SSAO inhibition to the biological effects of these drugs
remains unde®ned. More recently some potentially more
selective SSAO inhibitors like B24 (3,5-ethoxy-4-aminomethylpyridine) and MDL72274 have been developed
(Lyles, 1996).
SSAO protein sequences reveal conserved
motifs
In mammals the SSAO ®eld has been hampered by the fact
that the molecular identity of the enzymes has not been
known. Instead, the SSAO activity has been determined
merely by measuring enzymatic AO activity in cells,
tissues and body ¯uids. Gratifyingly, during recent years,
several SSAO species have been cloned from humans and
rodents, which helps to ascribe certain functions to
individual molecules. Determination of the SSAO
cDNAs has also started to unify the chaotic descriptive
nomenclature in the ®eld.
Most SSAOs are dimeric glycoproteins with molecular
masses of 140±180 kDa (Klinman and Mu, 1994; Lyles,
1996). SSAOs contain two atoms of copper per dimer.
Their molecular characterization started with isolation of
the molecules based on the physicochemical properties
and AO activity. Early on, profound interest has been in
the determination of chemical identity of the prosthetic
groups of these enzymes (Klinman, 1996). Initially,
pyrroloquinoline quinone was errorneously reported to
be the redox cofactor associated with bovine serum SSAO
(Lobenstein-Verbeek et al., 1984). When proteolytic
products of the active site peptides were ®nally obtained
in suf®cient quantities, an amino acid sequence LNXPY
was obtained and the unknown amino acid was identi®ed
as 6-hydroxydopa (2,4,5-trihydroxyphenylalanine quinone
or TPQ) (Janes et al., 1990; Klinman, 1996). TPQ is
generated from an intrinsic tyrosine of the molecule by a
self-processing event that only requires bound copper ion
and molecular oxygen (Mu et al., 1992).
Currently full-length cDNA sequences are available
from seven mammalian SSAOs. The ®rst monoamine
SSAO enzyme has been cloned from bovine liver and
encodes the bovine serum amine oxidase (BSAO) (Mu
et al., 1992). A human SSAO was then cloned by two
independent groups under two different names, human
placental amine oxidase (Zhang and McIntire, 1996) and
vascular adhesion protein-1 (VAP-1; see below; Smith
et al., 1998). Later, a retina-speci®c SSAO with alternatively spliced variants (designed RAO) (Imamura et al.,
1997, 1998) and another SSAO, which is apparently a
pseudogene (Cronin et al., 1998), have been reported. In
fact, these three human SSAOs were the only ones
identi®ed from the recently published sequence of the
human genome, and they all cluster in the long arm of
chromosome 17. The mouse and rat homologues of human
VAP-1 have also been cloned (Morris et al., 1997; Bono
et al., 1998a,b; Moldes et al., 1999). In addition, the
sequences of human and rat amiloride binding proteins
(Lingueglia et al., 1993) are known (see above). In bovine
tissues, there is genetic evidence for at least three SSAOs
(Hùgdall et al., 1998), suggesting that these enzymes form
a multimember family. The degree of amino acid identity
among the enzymes isolated from the same cellular origin
is high between the species. In contrast, intra- and
extracellular enzymes within one species differ more in
sequence.
Most SSAOs contain the conserved signature motif
(hydrophobic residue-Asn-Topa-Asp/Glu-Tyr) at the active site of the enzyme (Klinman and Mu, 1994; Salminen
et al., 1998). Tyrosine is always a precursor for the topa in
the sequence (Mu et al., 1992). There are three conserved
histidines in SSAOs, which coordinate the copper atoms.
One His-X-His motif ~50 residues C-terminal from the
cofactor and another histidine 20±30 residues toward the
N-terminus from the cofactor are conserved in all alignments, despite the variations in the length of whole
sequences. Moreover, a conserved Asp residue ~100
residues N-terminal from the topa is important, since it
serves as a catalytic base in the reductive half-reaction (see
below). The most important structural motifs of SSAOs
are illustrated in Figure 1B.
Initial sequence analyses predicted the presence of a
secretion signal in all SSAOs, including placental AO.
Therefore, the mechanism by which these enzymes were
inserted into the plasma membrane remained unknown for
a long time. Finally, epitope-tagged recombinant proteins
Fig. 1. (A) The classi®cation of AOs. NA, noradrenaline; DA, dopamine; A, adrenaline; b-PEA, b-phenylethylamine; trypt, tryptamine, ECM,
extracellular matrix; AOC, amine oxidase, copper-dependent. (B) The conserved motifs of SSAOs. In the N-terminus either a secretion signal or a
transmembrane segment is found. The characteristic positions of the catalytic base, copper-coordinating histidines and the four amino acids-long
sequence containing the tyrosine, which is modi®ed to TPQ, as an SSAO sequence signature are shown. The line above the SSAO molecule illustrates
the approximate amino acid positions of each important motif (the overall length of SSAO varies and hence the numbers are only approximations).
(C) An overall fold of the catalytically active domain (D4) of a human SSAO (VAP-1). The monomers are coloured red and blue. The inset shows a
closer view of the active site. The important active site residues are shown under the transparent surface. His520, His522 and His684 (blue) bind to
the copper ion (yellow). The other highlighted residues are TPQ, Tyr372, Tyr384 and Asp386 (red). Figure 1C is by the courtesy of Dr Tiina
Ê bo Akademi University, Turku, Finland.
Salminen, A
3895
S.Jalkanen and M.Salmi
revealed that, at least in VAP-1, the predicted secretion
signal is, in fact, a transmembrane segment (Smith et al.,
3896
1998). Hence, VAP-1 is a type 2 transmembrane protein
with a very short (4 amino acids) N-terminal cytoplasmic
Cell surface monoamine oxidases
tail, a single transmembrane segment and a large
extracellular segment.
A few SSAOs from bacteria have been puri®ed for
crystallography. A well characterized Escherichia coli
SSAO contains four domains and displays a mushroomlike shape (Parsons et al., 1995). A 400 amino acids-long
C-terminal b-sandwich domain contains the active site and
forms much of the dimer interface. Notably, the
b-sandwich core is a novel protein fold. The three other
a/b-domains are shorter (~100 amino acids each). The
dimeric structure is held together by connection, with
covalent and non-covalent interactions, of a pair of
b-hairpin turns. The active sites are deeply buried within
the molecule, but the only notable changes in structures
between inactive (in the absence of substrate) and active
enzyme are differences in copper coordination geometry
and the position and interactions of TPQ within the active
centre. Molecular modelling suggests that VAP-1 is also a
mushroom-shaped molecule (Figure 1C) (Salminen et al.,
1998).
Semicarbazide-sensitive amine oxidase exists in soluble
form(s) as well as in a membrane-bound form (Lyles,
1996). There has been considerable disagreement as to
whether the soluble form is a product of a different gene or
a cleavage product of the transmembrane form of SSAO.
In man there is evidence that the soluble enzyme is formed
by a proteolytic cleavage of the membrane-bound molecule, since the N-terminal sequence of the soluble form
isolated from the serum is identical to the membrane distal
sequence of the VAP-1. In fact, in man most, if not all, AO
activity in serum is mediated by soluble SSAO, which was
identi®ed as VAP-1 (KurkijaÈrvi et al., 2000). In other
animals, the situation may well be different, since kinetic
data suggest the existence of two different soluble SSAO
enzymes in sheep (Elliott et al., 1992; Boomsma et al.,
2000). Furthermore, the BSAO gene encoding a soluble
form of SSAO in bovine has been cloned (Mu et al., 1994).
The deamination reaction
All SSAOs catalyse oxidative deamination of primary
amines in a reaction: R-CH2-NH2 + O2 + H2O®RCHO + H2O2 + NH3 (reviewed in Klinman and Mu,
1994; Wilmot et al., 1999). The kinetic reaction consists
of two half-reactions (Figure 2A). First, the enzyme is
reduced by the substrate with simultaneous release of the
corresponding aldehyde. In the second part, the enzyme is
reoxidated by molecular oxygen with concomitant release
of hydrogen peroxide and ammonium.
Reductive inactivation experiments showed that the
substrate was trapped in a covalent bond to the enzyme
(Hartmann and Klinman, 1987). Nowadays there is direct
evidence that the reductive half-reaction involves sequential formation of multiple transition stages during which a
transient but covalent Schiff base is formed between the
enzyme and the substrate before the product aldehyde is
released (Dooley et al., 1991; Hartmann et al., 1993)
(Figure 2A). During the oxidative half-reaction, the
reduced cofactor recycles back to its oxidized TPQ form.
During this process hydrogen peroxide and ammonia are
released.
It is generally agreed that SSAOs only accept primary
amines as substrates, although there may be exceptions to
this rule (Yu and Davis, 1990). Nevertheless, there appears
to be wide variation among the preferred substrates in
different species (Lyles, 1996). Benzylamine, an arti®cial
amine, is the preferred substrate for most, if not all,
SSAOs. In addition, in humans methylamine and allylamine are accepted as SSAO substrates, but in many
rodents tyramine, tryptamine, histamine and b-phenylethylamine are also oxidatively deaminated by SSAOs.
Therefore, some caution must be exerted in generalizing
results with SSAOs obtained from different species.
SSAOs and catabolism of biogenic amines
Human SSAO can use xenobiotic amines like allylamine
as substrates (Lyles, 1996). During this reaction, these
compounds are converted to considerably more toxic
products (like acrolein from allylamine; Boor et al., 1990)
than the relatively harmless substrates themselves. Hence,
SSAO reactions may account for atherosclerotic lesions
seen in animals exposed to these amines.
Also, at least two endogenously formed amines can
serve as SSAO substrates. Thus, oxidation of methylamine,
which is formed during degradation of sarcosine, creatinine and adrenaline, results in formation of formaldehyde
(McEwen and Harrison, 1965). On the other hand, the end
products of SSAO-catalysed deamination of aminoacetone, which is derived from metabolism of glycine
and threonine, is methylglyoxal (Lyles and Chalmers,
1992). Again, the resulting aldehydes are much more toxic
than the parent compounds (Yu and Zuo, 1996) and the
physiological signi®cance of these reactions remains
unknown.
All products of the SSAO-catalysed reaction are
biologically active. Probably the most interesting of the
end-products is hydrogen peroxide. This reactive oxygen
species is toxic at high concentration. However, at lower
concentrations it is becoming increasingly recognized as a
signal-transducing molecule (Finkel, 1998; Kunsch and
Medford, 1999; Bogdan et al., 2000). Thus, hydrogen
Fig. 2. The SSAO reaction and leukocyte extravasation. (A) The catalytic reaction of SSAO. In the reductive half-reaction (1±4) the primary amine
group interacts with the TPQ of the enzyme. Then a proton is abstracted by the active-site base (aspartate) and, through a carbanionic intermediate, a
product Schiff base is formed. Thereafter, hydrolysis occurs, the product aldehyde is released and the reduced cofactor is left attached to enzyme
mainly in an aminoquinol-Cu2+ form. In the oxidative half-reaction (5, 6, 1) the reduced enzyme is recycled to the resting state via an iminoquinol
intermediate in a copper- and molecular oxygen-dependent reaction. During this half-reaction, hydrogen peroxide and ammonia are released. (B) The
leukocyte extravasation cascade. The different steps of the adhesion cascade and the involvement of VAP-1 are shown. (C) The oligosaccahride
modi®cations of VAP-1 (purple extensions) can bind to an unknown lectin-like molecule (yellow) on lymphocytes. Alternatively, when endothelial
VAP-1 uses a lymphocyte surface amine as a substrate, the catalytic reaction results in the formation of a transient covalent bond (step 3 in A)
between the two cell types. This enzymatic reaction seems to be involved in the binding during the rolling step. The oligosaccharide and Schiff-base
mediated bindings can involve separate molecules on the lymphocyte surface or, if the lectin-type lymphocyte surface molecule also presents the
amine to VAP-1, the same molecule may be used in both steps.
3897
S.Jalkanen and M.Salmi
peroxide known to regulate the function of transcription
factors, like NF-kB, and hence expression of many genes
(including chemokines, adhesion molecules, cytokines and
metalloproteinases). In the vascular wall hydrogen peroxide regulates proliferation and adhesive properties of both
endothelial cells and smooth muscle cells.
Functions of membrane-bound SSAO
The biological role of SSAOs (reviewed in Klinman and
Mu, 1994), with the exception of lysyl oxidase, has
remained enigmatic for decades in mammals. In bacteria
and yeast, the SSAO reaction provides these microbes with
a source of nitrogen (and carbon) when growing in the
presence of various amines. In plants, on the other hand,
the hydrogen peroxide released from the SSAO-catalysed
reaction is used for wound healing. In mammals, the
speculative functions of SSAOs have focused on their role
in metabolism of exogenous or endogenous amines (see
above), although the signi®cance of this reaction is far
from clear.
SSAOs are widely expressed in mammals (Lewinsohn,
1984; Salmi et al., 1993; Lyles, 1996; Jaakkola et al.,
1999). Based on enzymatic analyses, and con®rmed with
stainings of monoclonal anti-VAP-1 mAbs, the most
prominent synthesis takes place in smooth muscle (both
vascular and non-vascular) and adipocytes, but endothelial
cells and follicular dendritic cells are also positive for
VAP-1. In contrast, leukocytes, epithelial and ®broblastoid
cells are completely devoid of VAP-1. Notably, SSAO is
also absent from brain (except microvessels). In humans,
VAP-1 is also absent from chondrocytes and odontoblasts,
which have been reported to display SSAO activity in
other animals.
Quite recently, two totally new and different functions
for SSAOs have been proposed: they seem to be involved
in the regulation of glucose metabolism in adipose cells
and in the regulation of leukocyte traf®cking in endothelial
cells.
SSAOs and glucose metabolism
SSAOs account for ~1% of total adipocyte membrane
proteins, and its level is upregulated concordantly with
adipocyte differentiation (Morris et al., 1997;
Enrique-TarancoÂn et al., 1998; Moldes et al., 1999).
Thus, preadipocytes are practically SSAO negative,
whereas mature adipocytes express high levels of SSAO
protein and activity, which co-localizes with GLUT4
vesicles. When an arti®cial SSAO substrate benzylamine
is provided to mature adipocytes, glucose uptake is
signi®cantly enchanced via a GLUT4-dependent mechanism. This metabolic effect apparently depends on hydrogen peroxide formed, which may regulate traf®cking of
the glucose transporter GLUT4 to the plasma membrane.
The effect of SSAO activity on glucose metabolism and on
stimulation of tyrosine phosphorylation of insulin receptor
substrate (IRS) proteins and on activity of phosphatidylinositol-3-kinase has earlier only been seen in the presence
of vanadate, a potent phosphatase inhibitor
(Enrique-TarancoÂn et al., 1998, 2000). However, very
recent evidence shows that glucose transport is increased
in human adipocytes in the presence of SSAO substrates
3898
alone, which makes the biological signi®cance of these
observations even more evident (Morin et al., 2001).
SSAO/VAP-1 acts as an endothelial
adhesion molecule for leukocytes
Another line of investigation has surprisingly identi®ed an
SSAO as an adhesion molecule involved in leukocyte
traf®cking. This process consists of a multistep adhesion
cascade (Springer, 1994; Butcher and Picker, 1996; Salmi
and Jalkanen, 1997). Blood-borne leukocytes ®rst reversibly tether to and roll on the endothelial cells under
conditions of blood ¯ow. If they receive appropriate
activation signals, they can then ®rmly adhere to the vessel
wall and transmigrate into the tissue. Multiple adhesion
and activation molecules on leukocyte and endothelial cell
side regulate the molecular execution of this orchestrated
process (Figure 2B).
Using a mAb-based approach, an apparently novel
endothelial molecule designated VAP-1 was reported. It
mediates leukocyte binding in adhesion assays under shear
(Salmi and Jalkanen, 1992). VAP-1 is constitutively
present in intracellular granules within endothelial cells
(Salmi et al., 1993). However, with in vivo in¯ammation
models, VAP-1 seems to be translocated lumenally from
intracellular storage granules only upon elicition of
in¯ammation (Jaakkola et al., 2000). VAP-1 mediates
leukocyte subtype-speci®c adhesion (Salmi et al., 1997).
Among mononuclear cells, the VAP-1-dependent pathway
is important for binding of CD8-positive T killer cells and
natural killer cells, but not of B cells, T helper cells or
monocytes. VAP-1 is also present on sinusoidal endothelial cells in liver, which is a unique cell type supporting
leukocyte traf®cking to this large immunocompetent organ
(McNab et al., 1996). Sinusoidal VAP-1 also supports
lymphocyte adhesion in various adhesion assays (McNab
et al., 1996; Yoong et al., 1998). In animal studies, antiVAP mAbs ef®ciently inhibit (~70%) granulocyte extravasation into areas of acute in¯ammation (Tohka et al.,
2001). Finally, in intravital microscopy, the VAP-1
molecule ®rst seems to be important for the rolling
phase and most likely later at the transmigration step of
leukocyte extravasation (Tohka et al., 2001).
When VAP-1 was cloned, it revealed surprising
sequence identity to SSAOs (Smith et al., 1998).
Experiments with recombinant VAP-1 revealed that it
indeed possessed MAO activity with benzylamine and
methylamine being the preferred substrates. Very recently,
evidence has been obtained that the SSAO-catalysed
enzymatic reaction per se may be important for leukocyte
adhesion (Salmi et al., 2001). In parallel plate ¯ow
chamber analyses, VAP-1-positive endothelial cells support lymphocyte rolling and ®rm adhesion in a VAP-1dependent manner. Although the anti-VAP-1 mAbs inhibit
lymphocyte rolling, they do not inhibit the SSAO activity
of this dual function molecule. Most intriguingly, the
inclusion of chemical SSAO inhibitors speci®cally abrogated ~50% of lymphocyte rolling and ®rm adhesion
under physiological ¯ow. In contrast, inclusion of soluble
reaction products (hydrogen peroxide or benzaldehyde) to
the system did not inhibit lymphocyte±endothelial cell
interactions. Finally, provision of additional soluble SSAO
substrate for the reaction surprisingly inhibited rather than
Cell surface monoamine oxidases
augmented lymphocyte adherence. These data suggested
that a transient covalent bond may be formed between the
enzyme (endothelial VAP-1) and substrate during the
multistep extravasation cascade. Notably, a lymphoid cellsurface bound rather than a soluble amine had to be
postulated to be the SSAO substrate in this case to explain
the observations. This hypothesis was further substantiated
by the ®nding that a synthetic peptide can ®t into the
surface groove of VAP-1 and indeed modulate the SSAO
activity of VAP-1 by binding to the catalytic cavity (Salmi
et al., 2001). Thus, it is possible that surface bound amines
(like N-termini of proteins, NH2-containing amino acid
side chains, amino sugars etc.) may be SSAO substrates in
addition to soluble amines. Furthermore, this SSAO
function suggests a new way in which an endothelial
ecto-enzyme can directly regulate the leukocyte extravasation. Moreover, these data suggest that VAP-1 is a
dual function molecule: it binds lymphocytes via an antiVAP-1 mAb-de®ned epitope and subsequently utilizes the
catalytic reaction between a surface-bound amine and
VAP-1 to transiently link the interacting cells under
physiological ¯ow conditions. After the Schiff base step of
the enzymatic reaction (Figure 2A, step 3) has rapidly
turned over, the VAP-1-catalysed reaction may further
contribute to cross-linking if the protein-bound aldehydes
formed on lymphocytes interact with molecules on
endothelial cell surface.
Biological functions for soluble SSAOs
For the soluble SSAO at least two functions have so far
been proposed. First, increased levels of soluble serum
SSAO are found in speci®c diseases, most notably in
certain liver disorders and in diabetes (Garpenstrand et al.,
1999; KurkijaÈrvi et al., 2000). In diabetes there is evidence
that SSAO activity may cause the atherogenic lesions
typical of this disorder by catalysing extensive formation
of aldhehydes and reactive oxygen species from soluble
substrates (Yu and Deng, 1998). It may also be involved in
the production of non-enzymatic addition of oligosaccharides to proteins during formation of advanced glycosylation end products, typical of diabetic lesions (Yu and Zuo,
1993, 1997). On the other hand, soluble SSAO has been
shown to modulate lymphocyte adhesion to endothelial
cells (KurkijaÈrvi et al., 1998), presumably by triggering
positive signals on the lymphocyte.
Lysyl oxidaseÐa special case
Lysyl oxidase is unique amongst the SSAO family of
enzymes (Smith-Mungo and Kagan, 1998). Although it is
sensitive to SSAO inhibitors, it is considerably smaller
than the other SSAOs, its primary sequence lacks certain
critical motifs (e.g. the copper-coordinating histidines) and
its cofactor appears to be lysine tyrosylquinone rather
than TPQ (Wang et al., 1997). In fact, its classi®cation
is constantly under dispute. This molecule has a well
established role in cross-linking collagen and elastin
during the formation of extracellular matrix. Intriguingly, epsilon amino groups of lysine are known
substrates for this SSAO (Kagan et al., 1984), and it
exerts its function through covalent cross-linking of two
molecules (which then proceeds to a permanent linkage by
autocondensation). Hence, the molecular mechanism of
this enzyme is notably comparable to that proposed for
VAP-1 in leukocyte extravasation (see above).
Conclusions
SSAOs have been characterized by enzymatic means for
decades in serum as well as in multiple cell and tissue
types in various species. Based on this work, we currently
have a detailed understanding of the kinetic reaction these
enzymes catalyse. During the last 10 years, molecular
identi®cation of distinct SSAO species by molecular
cloning has opened new avenues for understanding the
biological functions of these abundant enzymes. One of
the most compelling questions at the moment is the nature
of physiological SSAO substrates. Moreover, there is now
evidence emerging that the function of SSAO may be
dependent on the cell type on which they are expressed. In
addition to functions connected to the catabolism of exoand endogenous biogenic amines, these enzymes may play
other roles as well. Notably, the possible functions of
SSAO end products in glucose metabolism and SSAOdependent Schiff-base formation in leukocyte traf®cking
are just two exciting examples of how the ectoenzymes
may regulate signalling and adhesion processes. SSAO
transgenic and knock-out animals have been generated,
and are currently under analysis. This should provide clear
answers to the questions of how individual SSAO
molecules contribute to various physiological and pathological functions in vascular biology and related areas.
Acknowledgements
We are indebted to Prof. J.Klinman, Prof. K.Tipton and Dr Gennady
Yegutkin for advice and critical reading of this manuscript. The
contribution of Prof. Klinman and Dr Diana Wertz in drawing
Figure 2A is especially highly appreciated. The enzymatic characterization of SSAOs in multiple species has been described in hundreds of
reports during the last decades, and we have only been able to refer to a
minor fraction of these papers in this short review. The help of Mrs Anne
Sovikoski-Georgieva in preparing the manuscript is acknowledged. The
original VAP-1/SSAO work has been supported by the Finnish Academy,
the European Union (QLG7-CT-1999±00295), the Sigrid Juselius
Foundation, the Finnish Cultural Foundation and the Finnish Cancer
Union.
References
Barbry,P. et al. (1990) Human kidney amiloride-binding protein: cDNA
structure and functional expression. Proc. Natl Acad. Sci. USA, 87,
7347±7351.
Bogdan,C., RoÈllinghoff,M. and Diefenbach,A. (2000) Reactive oxygen
and reactive nitrogen intermediates in innate and speci®c immunity.
Curr. Opin. Immunol., 12, 64±76.
Bono,P., Salmi,M., Smith,D.J. and Jalkanen,S. (1998a) Cloning and
characterization of mouse vascular adhesion protein-1 reveals a novel
molecule with enzymatic activity. J. Immunol., 160, 5563±5571.
Bono,P., Salmi,M., Smith,D.J., LeppaÈnen,I., Horelli-Kuitunen,N.,
Palotie,A.
and
Jalkanen,S.
(1998b)
Isolation,
structural
characterization and chromosomal mapping of the mouse vascular
adhesion protein-1 gene and promoter. J. Immunol., 161, 2953±2960.
Boomsma,F., van Dijk,J., Bhaggoe,U.M., Bouhuizen,A.M. and van den
Meiracker,A.H. (2000) Variation in semicarbazide-sensitive amine
oxidase activity in plasma and tissues of mammals. Comp. Biochem.
Physiol. C Toxicol. Pharmacol., 126, 69±78.
Boor,P.J., Hysmith,R.M. and Sanduja,R. (1990) A role for a new
vascular enzyme in the metabolism of xenobiotic amines. Circ. Res.,
66, 249±252.
3899
S.Jalkanen and M.Salmi
Buffoni,F. (1966) Histaminase and related amine oxidases. Pharmacol.
Rev., 18, 1163±1199.
Butcher,E.C. and Picker,L.J. (1996) Lymphocyte homing and
homeostasis. Science, 272, 60±66.
Cronin,C.N., Zhang,X., Thompson,D.A. and McIntire,W.S. (1998)
cDNA cloning of two splice variants of a human copper-containing
monoamine oxidase pseudogene containing a dimeric Alu repeat
sequence. Gene, 220, 71±76.
Dooley,D.M.,
McGuirl,M.A.,
Brown,D.E.,
Turowski,P.N.,
McIntire,W.S. and Knowles,P.F. (1991) A Cu(I)-semiquinone state
in substrate-reduced amine oxidases. Nature, 349, 262±264.
Elliott,J., Callingham,B.A. and Sharman,D.F. (1992) Amine oxidase
enzymes of sheep blood vessels and blood plasma: a comparison of
their properties. Comp. Biochem. Physiol. C, 102, 83±89.
Enrique-TarancoÂn,G. et al. (1998) Role of semicarbazide-sensitive
amine oxidase on glucose transport and GLUT4 recruitment to the
cell surface in adipose cells. J. Biol. Chem., 273, 8025±8032.
Enrique-TarancoÂn,G. et al. (2000) Substrates of semicarbazide-sensitive
amine oxidase co-operate with vanadate to stimulate tyrosine
phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells.
Biochem. J., 350, 171±180.
Finkel,T. (1998) Oxygen radicals and signaling. Curr. Opin. Cell Biol.,
10, 248±253.
Garpenstrand,H.,
Ekblom,J.,
BaÈcklund,L.B.,
Oreland,L.
and
Rosenqvist,U. (1999) Elevated plasma semicarbazide-sensitive
amine oxidase (SSAO) activity in Type 2 diabetes mellitus
complicated by retinopathy. Diabet. Med., 16, 514±521.
Goding,J.W. and Howard,M.C. (1998) Ecto-enzymes of lymphoid cells.
Immunol. Rev., 161, 5±10.
Hartmann,C. and Klinman,J.P. (1987) Reductive trapping of substrate to
bovine plasma amine oxidase [published erratum appears in J. Biol.
Chem., 1987, 262, 4427]. J. Biol. Chem., 262, 962±965.
Hartmann,C., Brzovic,P. and Klinman,J.P. (1993) Spectroscopic
detection of chemical intermediates in the reaction of parasubstituted benzylamines with bovine serum amine oxidase.
Biochemistry, 32, 2234±2241.
Hùgdall,E.V.S., Houen,G., Borre,M., Bundgaard,J.R., Larsson,L.-I. and
Vuust,J. (1998) Structure and tissue-speci®c expression of genes
encoding bovine copper amine oxidases. Eur. J. Biochem., 251,
320±328.
Imamura,Y., Kubota,R., Wang,Y., Asakawa,S., Kudoh,J., Mashima,Y.,
Oguchi,Y. and Shimizu,N. (1997) Human retina-speci®c amine
oxidase (RAO): cDNA cloning, tissue expression and chromosomal
mapping. Genomics, 40, 277±283.
Imamura,Y., Noda,S., Mashima,Y., Kudoh,J., Oguchi,Y. and Shimizu,N.
(1998) Human retina-speci®c amine oxidase: genomic structure of the
gene (AOC2), alternatively spliced variant and mRNA expression in
retina. Genomics, 51, 293±298.
Jaakkola,K. et al. (1999) Human vascular adhesion protein-1 in smooth
muscle cells. Am. J. Pathol., 155, 1953±1965.
Jaakkola,K. et al. (2000) In vivo detection of vascular adhesion protein-1
in experimental in¯ammation. Am. J. Pathol., 157, 463±471.
Janes,S.M., Mu,D., Wemmer,D., Smith,A.J., Kaur,S., Maltby,D.,
Burlingame,A.L. and Klinman,J.P. (1990) A new redox cofactor in
eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum
amine oxidase. Science, 248, 981±987.
Kagan,H.M., Williams,M.A., Williamson,P.R. and Anderson,J.M.
(1984) In¯uence of sequence and charge on the speci®city of lysyl
oxidase toward protein and synthetic peptide substrates. J. Biol.
Chem., 259, 11203±11207.
Klinman,J.P. (1996) New quinocofactors in eukaryotes. J. Biol. Chem.,
271, 27189±27192.
Klinman,J.P. and Mu,D. (1994) Quinoenzymes in biology. Annu. Rev.
Biochem., 63, 299±344.
Kunsch,C. and Medford,R.M. (1999) Oxidative stress as a regulator of
gene expression in the vasculature. Circ. Res., 85, 753±766.
KurkijaÈrvi,R., Adams,D.H., Leino,R., MoÈttoÈnen,T., Jalkanen,S. and
Salmi,M. (1998) Circulating form of human vascular adhesion
protein-1 (VAP-1): increased serum levels in in¯ammatory liver
diseases. J. Immunol., 161, 1549±1557.
KurkijaÈrvi,R., Yegutkin,G.G., Gunson,B.K., Jalkanen,S., Salmi,M. and
Adams,D.H. (2000) Circulating soluble vascular adhesion protein 1
accounts for the increased serum monoamine oxidase activity in
chronic liver disease. Gastroenterology, 119, 1096±1103.
Lewinsohn,R. (1984) Mammalian monoamine-oxidizing enzymes, with
3900
special reference to benzylamine oxidase in human tissues. Braz. J.
Med. Biol. Res., 17, 223±256.
Lingueglia,E., Renard,S., Voilley,N., Waldmann,R., Chassande,O.,
Lazdunski,M. and Barbry,P. (1993) Molecular cloning and
functional expression of different molecular forms of rat amiloridebinding proteins. Eur. J. Biochem., 216, 679±687.
Lobenstein-Verbeek,C.L., Jongejan,J.A., Frank,J. and Duine,J.A. (1984)
Bovine serum amine oxidase: a mammalian enzyme having covalently
bound PQQ as prosthetic group. FEBS Lett., 170, 305±309.
Lyles,G.A. (1996) Mammalian plasma and tissue-bound semicarbazidesensitive amine oxidases: biochemical, pharmacological and
toxicological aspects. Int. J. Biochem. Cell Biol., 28, 259±274.
Lyles,G.A. and Chalmers,J. (1992) The metabolism of aminoacetone to
methylglyoxal by semicarbazide-sensitive amine oxidase in human
umbilical artery. Biochem. Pharmacol., 43, 1409±1414.
McEwen,C.M.,Jr and Harrison,D.C. (1965) Abnormalities of serum
monoamine oxidase in chronic congestive heart failure. J. Lab. Clin.
Med., 65, 546±559.
McNab,G., Reeves,J.L., Salmi,M., Hubscher,S., Jalkanen,S. and
Adams,D.H. (1996) Vascular adhesion protein 1 mediates binding of
T cells to human hepatic endothelium. Gastroenterology, 110,
522±528.
Moldes,M., FeÁve,B. and Pairault,J. (1999) Molecular cloning of a major
mRNA species in murine 3T3 adipocyte lineage. Differentiationdependent expression, regulation and identi®cation as semicarbazidesensitive amine oxidase. J. Biol. Chem., 274, 9515±9523.
Morin,N. et al. (2001) Semicarbazide-sensitive amine oxidase substrates
stimulate glucose transport and inhibit lipolysis in human adipocytes.
J. Pharmacol. Exp. Ther., 297, 563±572.
Morris,N.J., Ducret,A., Aebersold,R., Ross,S.A., Keller,S.R. and
Lienhard,G.E. (1997) Membrane amine oxidase cloning and
identi®cation as a major protein in the adipocyte plasma membrane.
J. Biol. Chem., 272, 9388±9392.
Mu,D., Janes,S.M., Smith,A.J., Brown,D.E., Dooley,D.M. and
Klinman,J.P. (1992) Tyrosine codon corresponds to topa quinone at
the active site of copper amine oxidases. J. Biol. Chem., 267,
7979±7982.
Mu,D., Medzihradszky,K.F., Adams,G.W., Mayer,P., Hines,W.M.,
Burlingame,A.L., Smith,A.J., Cai,D. and Klinman,J.P. (1994)
Primary structures for a mammalian cellular and serum copper
amine oxidase. J. Biol. Chem., 269, 9926±9932.
Novotny,W.F., Chassande,O., Baker,M., Lazdunski,M. and Barbry,P.
(1994) Diamine oxidase is the amiloride-binding protein and is
inhibited by amiloride analogues. J. Biol. Chem., 269, 9921±9925.
Parsons,M.R., Convery,M.A., Wilmot,C.M., Yadav,K.D.S., Blakeley,V.,
Corner,A.S., Phillips,S.E.V., McPherson,M.J. and Knowles,P.F.
(1995) Crystal structure of a quinoenzyme: copper amine oxidase of
Ê resolution. Structure, 3, 1171±1184.
Escherichia coli at 2 A
Robinson-White,A., Baylin,S.B., Olivecrona,T. and Beaven,M.A. (1985)
Binding of diamine oxidase activity to rat and guinea pig
microvascular endothelial cells. Comparisons with lipoprotein lipase
binding. J. Clin. Invest., 76, 93±100.
Salmi,M. and Jalkanen,S. (1992) A 90-kilodalton endothelial cell
molecule mediating lymphocyte binding in humans. Science, 257,
1407±1409.
Salmi,M. and Jalkanen,S. (1997) How do lymphocytes know where to
go: current concepts and enigmas of lymphocyte homing. Adv.
Immunol., 64, 139±218.
Salmi,M., Kalimo,K. and Jalkanen,S. (1993) Induction and function of
vascular adhesion protein-1 at sites of in¯ammation. J. Exp. Med.,
178, 2255±2260.
Salmi,M., Tohka,S., Berg,E.L., Butcher,E.C. and Jalkanen,S. (1997)
Vascular adhesion protein 1 (VAP-1) mediates lymphocyte subtypespeci®c, selectin-independent recognition of vascular endothelium in
human lymph nodes. J. Exp. Med., 186, 589±600.
Salmi,M., Yegutkin,G., Lehvonen,R., Koskinen,K., Salminen,T. and
Jalkanen,S. (2001) A cell surface amine oxidase directly controls
lymphocyte migration. Immunity, 14, 265±276.
Salminen,T.A., Smith,D.J., Jalkanen,S. and Johnson,M.S. (1998)
Structural model of the catalytic domain of an enzyme with cell
adhesion activity: human vascular adhesion protein-1 (HVAP-1) D4
domain is an amine oxidase. Protein Eng., 11, 1195±1204.
Seiler,N. (1990) Polyamine metabolism. Digestion, 46, 319±330.
Shih,J.C., Chen,K. and Ridd,M.J. (1999) Monoamine oxidase: from
genes to behavior. Annu. Rev. Neurosci., 22, 197±217.
Smith,D.J., Salmi,M., Bono,P., Hellman,J., Leu,T. and Jalkanen,S.
Cell surface monoamine oxidases
(1998) Cloning of vascular adhesion protein-1 reveals a novel
multifunctional adhesion molecule. J. Exp. Med., 188, 17±27.
Smith-Mungo,L.I. and Kagan,H.M. (1998) Lysyl oxidase: properties,
regulation and multiple functions in biology. Matrix Biol., 16,
387±398.
Springer,T.A. (1994) Traf®c signals for lymphocyte recirculation and
leukocyte emigration: the multistep paradigm. Cell, 76, 301±314.
Tabor,C.W., Tabor,H. and Rosenthal,S.M. (1954) Puri®cation of amine
oxidase from beef plasma. J. Biol. Chem., 208, 645±661.
Tohka,S., Laukkanen,M.-L., Jalkanen,S. and Salmi,M. (2001) Vascular
adhesion protein 1 (VAP-1) functions as a molecular brake during
granulocyte rolling and mediates their recruitment in vivo. FASEB J.,
15, 373±382.
Wang,S.X., Nakamura,N., Mure,M., Klinman,J.P. and Sanders-Loehr,J.
(1997) Characterization of the native lysine tyrosylquinone cofactor in
lysyl oxidase by Raman spectroscopy. J. Biol. Chem., 272,
28841±28844.
Wilmot,C.M., Hajdu,J., McPherson,M.J., Knowles,P.F. and Phillips,S.E.
(1999) Visualization of dioxygen bound to copper during enzyme
catalysis. Science, 286, 1724±1728.
Yoong,K.F., McNab,G., HuÈbscher,S.G. and Adams,D.H. (1998)
Vascular adhesion protein-1 and ICAM-1 support the adhesion of
tumor-in®ltrating lymphocytes to tumor endothelium in human
hepatocellular carcinoma. J. Immunol., 160, 3978±3988.
Yu,P.H. and Davis,B.A. (1990) Some pharmacological implications of
MAO-mediated deamination of branched aliphatic amines: 2-propyl1-aminopentane and N-(2-propylpentyl)glycinamide as valproic acid
precursors. J. Neural Transm. Suppl., 32, 89±92.
Yu,P.H. and Deng,Y.L. (1998) Endogenous formaldehyde as a potential
factor of vulnerability of atherosclerosis: involvement of semicarbazide-sensitive amine oxidase-mediated methylamine turnover.
Atherosclerosis, 140, 357±363.
Yu,P.H. and Zuo,D.-M. (1993) Oxidative deamination of methylamine
by semicarbazide-sensitive amine oxidase leads to cytotoxic damage
in endothelial cells. Possible consequences for diabetes. Diabetes, 42,
594±603.
Yu,P.H. and Zuo,D.M. (1996) Formaldehyde produced endogenously via
deamination of methylamine. A potential risk factor for initiation of
endothelial injury. Atherosclerosis, 120, 189±197.
Yu,P.H. and Zuo,D.M. (1997) Aminoguanidine inhibits semicarbazidesensitive amine oxidase activity: implications for advanced glycation
and diabetic complications. Diabetologia, 40, 1243±1250.
Zhang,X. and McIntire,W.S. (1996) Cloning and sequencing of a coppercontaining, topa quinone-containing monoamine oxidase from human
placenta. Gene, 179, 279±286.
Received April 11, 2001; revised June 7, 2001;
accepted June 15, 2001
3901